ONT direct RNA sequencing of soybean under salt stress. The transcriptome complexity of cultivated soybean under salt stress was revealed by long-read sequencing on native mRNAs

Gene expression regulation is crucial for plant adaptation to adverse environmental conditions. Besides alternative splicing, poly(A) tail length and m6A modification have also been suggested to contribute to gene expression and regulation. However, the typical RNA sequencing approach using reverse-transcribed mRNAs cannot concurrently profile all these mRNA processing events. Here, we used Oxford Nanopore Technologies Direct RNA Sequencing (ONT DRS) to profile (epi-)transcriptomes of leaves and roots of cultivated soybean under short-term (1-h) salt stress, and found more than 89,586 novel transcript isoforms in addition to the 44,877 annotated isoforms. Specifically, we identified 102,191 alternative mRNA processing events and 1,216 fusion transcripts corresponding to 549 genomic regions. Interestingly, genes upregulated due to salt stress had longer poly(A) tail lengths and lower m6A modification ratios in salt-stressed roots compared to controls, and downregulated genes had shorter poly(A) tail lengths in roots. The negative correlation between poly(A) tail lengths and m6A modification levels in the transcripts of upregulated genes in roots indicated that these may be additional mechanisms to regulate gene expressions and mRNA stability in soybean experiencing salt stress. These results provide new dimensions for investigating the mechanisms of gene expression and mRNA regulation in soybean.